Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 57
Figure 2.48. Effects of self-absorption of anthracene on its emission
spectrum. A 1-cm 2 cuvette was used with right-angle observation.
Revised from [61].
rophores like fluorescein with a small Stokes shift are particularly
sensitive to concentration quenching.
High optical densities can distort the emission spectra
as well as the apparent intensities. For example, when rightangle
observation is used, the short-wavelength emission
bands of anthracene are selectively attenuated (Figure
2.48). This occurs because these shorter wavelengths are
absorbed by anthracene. Attenuation of the blue edge of the
emission is most pronounced for fluorophores that have significant
overlap of the absorption and emission spectra. Fluorophores
that display a large Stokes shift are less sensitive
to this phenomenon.
A dramatic effect of concentration can be seen with
fluorophores that display a small Stokes shift. Figure 2.49
shows a photograph of three bottles of rhodamine 6G on a
light box, with the concentration increasing from left to
right. The color changes from green to orange. This effect
is due to reabsorption of the shorter wavelength part of the
emission. The emission spectra shift dramatically to longer
wavelengths at higher concentrations.
2.12. COMMON ERRORS IN SAMPLE
PREPARATION
It is valuable to summarize some of the difficulties that can
be encountered with any given sample (Figure 2.50). The
sample can be too concentrated, in which case all the light
is absorbed at the surface facing the light source. In fact,
this is one of the more common errors. With highly absorbing
solutions and right-angle observations the signal levels
can be very low. Other problems are when the sample contains
a fluorescent impurity, or the detected light is contam-
Figure 2.49. Effect of concentrations on the color and emission spectra
of rhodamine 6G. The concentrations of R6G are 5 x 10 –6 , 1.6 x
10 –4 , and 5.7 x 10 –3 M. From [64].
inated by Rayleigh or Raman scatter. Sometimes the signal
may seem too noisy given the signal level. Intensity fluctuations
can be due to particles that drift through the laser
beam, and fluoresce or scatter the incident light.
Even if the fluorescence is strong, it is important to
consider the possibility of two or more fluorophores, that is,
an impure sample. Emission spectra are usually independent
of excitation wavelength. 65 Hence it is useful to determine
if the emission spectrum remains the same at different
excitation wavelengths.
One example of a mixture of fluorophores is shown in
Figure 2.51, which contains a mixture of coumarin 102
(C102) and coumarin 153 (C153). For a pure solution of
C102 the same emission spectrum is observed for excitation
at 360 and 420 nm (top). For a mixture of C102 and C153,
one finds an increased intensity above 500 nm for excitation
at 420 (bottom, dashed). This peak at 520 nm is due to
C153, which can be seen from its emission spectrum (dot-